The present invention relates to methods of extractively purifying and/or isolating antibiotics and/or antimicrobial agents. More particularly, the present invention relates to an inexpensive and efficient extractive method for purifying or isolating lipopeptide antibiotics.
An important class of antibiotics that inhibit gram-positive bacteria are the acidic lipopeptide antibiotics. Generally, acidic lipopeptide antibiotics consist of either a cyclic peptide core or a cyclic depsipeptide core acylated with a lipophilic fragment and have an isoelectric point of less than about pH 7.0. The lipophilic fragment, typically an unsaturated fatty acid, may be of varying length. Frequently, the antibiotic activity of lipopeptide antibiotics is related to the length of the lipophilic fragment.
Examples of acidic lipopeptide antibiotics include, but are not limited to, laspartomycin (Umezawa et al., U.S. Pat. No. 3,639,582; Naganawa et al., 1968, J Antibiot., 21, 55; Naganawa et al., 1970, J Antibiot., 23, 423), zaomycin (Kuroya, 1960, Antibiotics Ann., 194; Kuroya, JP 8150), crystallomycin (Gauze et al, 1957, Antibiotiki, 2, 9), aspartocin (Shay et al., 1960, Antibiotics Annual, 194; Hausman et al., 1964, Antimicrob. Ag. Chemother., 352; Hausman et al., 1969, J Antibiot., 22, 207; Martin et al., 1960, J Am. Chem. Soc., 2079), amphomycin (Bodanszky et. al., 1973, J Am. Chem. Soc., 95, 2352), glumamycin (Fujino et al., 1965, Bull. Chem. Soc. Jap., 38, 515), brevistin (Shoji et al., 1976, J Antibiotics, 29, 380), cerexin A (Shoji et al., 1976, J Antibiotics, 29, 1268), cerexin B (Shoji et al., 1976, J Antibiotics, 29, 1275) Antibiotic A-30912 (Hoehn et al., U.S. Pat. No. 5,039,789), Antibiotic A-1437 (Hammann et al., EP 0 629 636 B1; Lattrell et al., U.S. Pat. No. 5,629,288), Antibiotic A-54145 (Fukada et al., U.S. Pat. No. 5,039,789; Boeck et al., 1990, J Antibiotics, 43, 587), Antibiotic A-21978C (Debono et al., 1988, J. Antibiotics, 41, 1093) and tsushimycin (Shoji et. al., 1968, J Antibiot., 21, 439). See also Berdy, xe2x80x9cCRC Handbook of Antibiotic Compounds,xe2x80x9d Volume IV, Part 1, pages 313-327, CRC Press, Boca Raton, Fla., (1980); Korzybinski et al., xe2x80x9cAntibiotics-Origin Nature and Properties,xe2x80x9d Vol. 1, Pergamon Press, pp. 397-401 and 404-408, New York, N.Y. (1967).
The acidic lipopeptide antibiotics are typically active against Gram-positive microbes and constitute important therapeutics in the treatment of infections caused by these bacteria. However, conventional procedures used to isolate and purify acidic lipopeptide antibiotics from fermentation broths involve a number of extraction and chromatography steps, which are time consuming, labor intensive and expensive to carry out on a commercial scale. Thus, there is a need in the art for improved methods of isolating and/or purifying acidic lipopeptide antibiotics.
These and other needs are addressed by the present invention, which provides a rapid and inexpensive extractive method for purifying large quantities of lipopeptide antibiotics in high yield. Quite surprisingly, it has been discovered that acidic lipopeptide antibiotics such as laspartomycin, amphomycin and aspartocin, which have cyclic peptide nuclei and Antibiotic A-21978C, which has a cyclic depsipeptide nucleus, may under conditions of pH that are above the isoelectric point of the lipopeptide antibiotic and in the presence of divalent metal cations such as Ca+2, be directly extracted into water-immiscible organic solvents such as 1-butanol.
While not intending to be bound by any particular theory of operation, it is believed that acidic lipopeptide antibiotics form chelates with divalent metal cations such as, for example, Ca+2 that are stable under basic conditions and that are soluble in water-immiscible organic solvents such as 1-butanol. Under acidic conditions, the chelates are disrupted and acidic lipopeptide antibiotics may be extracted into aqueous solution at basic or near neutral pH. Thus, according to one embodiment of the invention, the method comprises contacting an aqueous composition comprising a lipopeptide antibiotic and a divalent metal cation and having a pH above the isoelectric point of the lipopeptide antibiotic with a water immiscible organic solvent, thereby extracting the lipopeptide antibiotic into organic solvent. Preferably, the pH of the aqueous composition is neutral or basic.
The lipopeptide antibiotic may then be extracted into aqueous solution from organic solvent by acidifying the organic solvent at a pH below the isoelectric point of the lipopeptide antibiotic followed by contacting the acidified organic solvent with an aqueous solution, which is at neutral or basic pH. The lipopeptide antibiotic, which now behaves like a conventional carboxylic acid, may be extracted back into organic solvent by acidifying the aqueous solution and extracting the aqueous solution with organic solvent. At this point, if necessary, the lipopeptide antibiotic may be further purified using extractive or chromatographic purification.
The extractive isolation methods of the current invention may be used to isolate and/or purify acidic lipopeptide antibiotics directly from fermentation or culture broths, either before or after removal of cells and/or cell debris and/or insoluble matter. Alternatively, the extractive isolation methods of the invention may be used in combination with conventional isolation and purification techniques. For example, an acidic lipopeptide antibiotic may be first precipitated from fermentation or culture medium and the antibiotic isolated and/or purified from the precipitate according to the extractive isolation methods of the invention. The methods of the current invention may be used to advantageously isolate and/or purify synthetic acidic lipopeptide antibiotics and/or derivatives such as the synthetic lipopeptide derivatives described, for example, in Debono et al., 1988, J Antibiotics, 41, 1093 and Lattrell et al., U.S. Pat. No. 5,629,288.
Thus, when used either alone or in combination with standard extraction and chromatographic techniques, the extractive methods of the invention allow for the isolation of acidic lipopeptide antibiotics in high yield and high purity with fewer steps than are required by conventional methods.
Reference will now be made in detail to preferred embodiments of the invention. While the invention will be described in conjunction with preferred embodiments, it should be understood that it is not intended to limit the invention to these preferred embodiments. To the contrary, it is intended to cover alternatives, modifications, and equivalents as may be included within the spirit and scope of the invention as defined by the appended claims.
The extractive purification methods of the invention may be used to rapidly and inexpensively isolate and/or purify virtually any acidic lipopeptide antibiotic. As used herein, xe2x80x9cacidic lipopeptide antibioticxe2x80x9d refers to those antibiotics that have a cyclic peptidic nucleus with a lipophilic fragment such as a fatty acid chain attached thereto and an isoelectric point of less than about pH 7.0. The cyclic peptidic nucleus may be a cyclic peptide or a cyclic depsipeptide. The lipophilic fragment may be attached directly to the nucleus or through a linker, which is typically peptidic.
Acidic lipopeptide antibiotics may be natural products, synthetic or semisynthetic. Acidic lipopeptide antibiotics may also be derivatives of natural or synthetic acidic lipopeptide antibiotics, provided that the derivatives include carboxyl groups which permit extractive isolation according to the principles of the current invention.
Exemplary acidic lipopeptide antibiotics which can be advantageously isolated and/or purified according to the extractive methods of the invention include, but are not limited to, laspartomycin (Umezawa et al., U.S. Pat. No. 3,639,582; Naganawa et al., 1968, J Antibiot., 21, 55; Naganawa et al, 1970, J Antibiot., 23, 423), zaomycin (Kuroya, 1960, Antibiotics Ann., 194; Kuroya, JP 8150), crystallomycin (Gauze et al., 1957, Antibiotiki, 2, 9), aspartocin (Shay et al., 1960, Antibiotics Annual, 194; Hausman et al., 1964, Antimicrob. Ag. Chemother., 352; Hausman et al., 1969, J Antibiot., 22, 207; Martin et al., 1960, J Am. Chem. Soc., 2079), amphomycin (Bodanszky et. al., 1973, J Am. Chem. Soc., 95, 2352), glumamycin (Fujino et al., 1965, Bull. Chem. Soc. Jap., 38, 515), brevistin (Shoji et al., 1976, J Antibiotics, 29, 380), cerexin A (Shoji et al., 1976, J Antibiotics, 29, 1268), cerexin B (Shoji et al., 1976, J Antibiotics, 29, 1275) Antibiotic A-30912 (Hoehn et al., U.S. Pat. No. 5,039,789), Antibiotic A-1437 (Hammann et al., EP 0 629 636 BI; Lattrell et al., U.S. Pat. No. 5,629,288), Antibiotic A-54145 (Fukada et al., U.S. Pat. No. 5,039,789; Boeck et al., 1990, J Antibiotics, 43, 587), Antibiotic A-21978C (Debono et al., 1988, J Antibiotics, 41, 1093) and tsushimycin (Shoji et. al., 1968, J Antibiot., 21, 439). See also Berdy, xe2x80x9cCRC Handbook of Antibiotic Compounds,xe2x80x9d Volume IV, Part 1, pages 313-327, CRC,Press, Boca Raton, Fla., (1980); Korzybinski et al., xe2x80x9cAntibiotics-Origin Nature and Properties,xe2x80x9d Vol. I, Pergamon Press, pp. 397-401 and 404-408, New York, N.Y. (1967).
Microorganisms that synthesize these various acidic lipopeptide antibiotics, as well as methods and conditions under which the microorganism may be cultured to provide the various lipopeptide antibiotics are well known in the art (see e.g., Umezawa et al., U.S. Pat. No. 3,639,582; Debono et. al., 1988, J Antibiotics 41: 1093; Shay et al., 1960, Antibiotics Annual 194; Hamill et al., U.S. Pat. No. 4,331,594; Hamill et al., U.S. Pat. No. 4,208,403; Hoehn et al., U.S. Pat. No. 4,024,245; Higgins et al., U.S. Pat. No. 4,024,246; Boeck et al., U.S. Pat. No. 4,288,549; Boeck et al., U.S. Pat. No. 4,994,270; Boeck, U.S. Pat. No. 4,977,083).
Those of skill in the art will appreciate that many acidic lipopeptide antibiotics are natural fermentation products comprising mixtures of isomeric compounds. The various natural product isomers differ in one or more respects, typically in the lengths, branching, and/or degree of saturation of their respective fatty acid side chains. In other instance, such as the semisynthetic lipopeptide antibiotics described in Debono et al., 1988, J Antibiotics, 41, 1093 and Lattrell et al., U.S. Pat. No. 5,629,288 or those instances where natural product mixtures have been separated or where fermentation or culture conditions are controlled such that a single type of molecule is produced, acidic lipopeptide antibiotic preparations will be xe2x80x9cpurexe2x80x9d with respect to the antibiotic molecule (i.e., it will not comprise a mixture of molecules). It should be understood that the extractive methods of the invention may be used to isolate acidic lipopeptide antibiotics regardless of whether they constitute a mixture of molecules or a single type of molecule. However the methods of the invention do not separate different molecules of a mixture of natural fermentation products from one another. Thus, if the producing microorganism synthesizes a mixture of acidic lipopeptide antibiotics, the extractive methods of the invention may be used to isolate the mixture from other contaminants and impurities.
Acidic lipopeptide antibiotics under normal conditions are not extracted into organic solvents, even polar organic solvents, from neutral or basic aqueous solutions. Thus, when a neutral or basic aqueous solution of a acidic lipopeptide antibiotic is washed or contacted with organic solvents, the lipopeptide antibiotic typically remains in the aqueous phase, as expected for a compound that contains multiple carboxyl groups.
It has been discovered that under specified conditions, acidic lipopeptide antibiotics may be extracted into organic solvents from neutral or basic aqueous solution, which enables easy separation from acidic impurities by simple extraction. As is well known by those of skill in the art, acidic lipopeptide antibiotics may be easily separated from neutral and basic impurities by converting carboxyl groups of the antibiotic to carboxylate groups (i.e., by treating acidic lipopeptide antibiotics with base), extracting the carboxylate into aqueous solution, converting the carboxylate groups back to carboxyl groups (i.e., by treating acidic lipopeptide antibiotics with acid), and extracting the carboxyl form into organic solvent. Thus, the method of the current invention, when used in conjunction with known methods of extractively purifying lipopeptide antibiotics, enables isolation of these compounds freed from acidic, basic and neutral impurities in good yield and high purity through simple extraction, which avoids the use of expensive and time consuming chromatography steps.
The conditions which permit an acidic lipopeptide antibiotic to be partitioned or extracted into organic solvents from aqueous solution under neutral or basic conditions are related to the pH and the presence of divalent metal cation such as Ca+2 in the aqueous solution containing the lipopeptide antibiotic. Generally, acidic lipopeptide antibiotics may be extracted into organic solvents from aqueous solutions that contain divalent metal ion, which are maintained at a pH above the isoelectric point of the antibiotics.
While not wishing to be bound by any particular theory of operation, it is believed that adjusting the pH of the solution above the isoelectric point of the acidic lipopeptide antibiotic ionizes the carboxyl groups. The carboxylate groups bind available divalent metal to form a stable divalent metal chelate of the antibiotic. The chelate, unlike the carboxylate anion of a acidic lipopeptide antibiotic, may be extracted into organic solvents from aqueous solution. Treating or washing organic solvents that contain the chelate of an acidic lipopeptide antibiotic with acid disrupts the chelate, thus providing the native acidic lipopeptide antibiotic.
Owing to this believed theory of operation, reference is made through the application to an xe2x80x9cacidic lipopeptide antibiotic chelate.xe2x80x9d However, it will be understood that this expression is being used merely for illustration and as a means for identifying a form of acidic lipopeptide antibiotic that can be extracted into organic solvent systems, and is not intended to be limiting in any way.
Acidic lipopeptide antibiotics may be isolated and/or purified according to the invention directly from fermentation and/or culture broth, either with or without prior removal of cell debris. Alternatively, acidic lipopeptide antibiotics may first be isolated by conventional means, such as by acidic precipitation, and the precipitate resuspended and isolated and/or purified according to the extractive methods of the invention. The methods of the invention may also be used to isolate and/or purify synthetic acidic lipopeptide antibiotics and/or derivatives thereof.
As discussed above, a divalent metal cation may be chelated by ionized caboxylate groups of the acidic lipopeptide antibiotic under certain conditions. Thus, prior to extraction, the pH of the aqueous solution comprising an acidic lipopeptide antibiotic should be sufficiently basic to ionize the carboxyl groups of the lipopeptide antibiotic. Typically, the pH of the aqueous solution is adjusted to at least a pH above the isoelectric point of the particular lipopeptide antibiotic being isolated. However, as the efficiency of the extraction is believed to depend on chelate formation, the pH of the aqueous solution containing the lipopeptide antibiotic is ideally adjusted to a value that is sufficiently basic to ionize all of the carboxyl groups of the lipopeptide antibiotic (i.e., at least about pH 5.0). Preferably, the pH of the aqueous solution is between about 7.0 and about 9.0, more preferably, between about 8.0 and about 9.0 and most preferably, between about 8.5 and about 9.0. Of course, if the acidic lipopeptide antibiotic is extracted directly from fermentation or culture broth, the pH of the broth may be sufficiently basic to render further adjustments unnecessary.
In order to form an acidic lipopeptide antibiotic chelate, the aqueous solution must include a divalent metal cation. Divalent metal cations that can form chelates with acidic lipopeptide antibiotics, which may be extracted into organic solvents according to the current invention include, but are not limited to, Ca+2, Mg+2, Zn30 2, Mn+2, Cu+2 and N+2. Preferred divalent metal cations include Ca+2, Mg+2 and Zn+2.
The amount or concentration of the divalent metal cation is not critical to success. However, since the method of the current invention is believed to operate by chelating carboxylate groups of the lipopeptide antibiotic, the molar concentration of divalent metal cation relative to the molar concentration of the acidic lipopeptide antibiotic carboxylate groups is at least about 0.5. Preferably, the molar ratio of divalent metal cation to carboxyl groups in the lipopeptide antibiotic is between about 4:1 to about 10:1. If the number of carboxyl groups in a particular acidic lipopeptide antibiotic is unknown, a desired divalent metal cation concentration and/or molar ratio may be readily determined empirically.
Divalent metal cation may be added to the aqueous solution by way of salts, and may be added before or after culturing or fermenting the producing strain, depending upon the requirement of the producing strain. The identity of the counter anion(s) is not critical; however, if the salt is added prior to culturing or fermenting the producing strain, a counter anion that negatively impacts the microorganism culture or fermentation broth should be avoided.
In many instances, the culture or fermentation broth and/or resuspended precipitate may contain a sufficient amount of divalent metal cation such that addition of further cation may be unnecessary. Whether the addition of further divalent metal cation is necessary may be determined by routine experimentation. Once the acidic lipopeptide antibiotic chelate has formed, it may be extracted into organic solvent by contacting or washing the aqueous solution comprising the chelate with organic solvent.
The organic solvent used to extract the acidic lipopeptide antibiotic chelate is not critical. However, it should satisfy two criteria: first, it should dissolve appreciable quantities of the acidic lipopeptide antibiotic chelate (i.e., the acidic lipopeptide antibiotic chelate should be more than sparingly soluble in the selected solvent system) and second, it should be at least partially immiscible with aqueous solutions (i.e., the aqueous solution and the organic solvent system should form two phases after mixing). Preferably, the organic solvent is a polar solvent in which the acidic lipopeptide antibiotic chelate is more than sparingly soluble, which is substantially immiscible with water. The organic solvent may be a pure solvent or a mixture of solvents. Suitable solvents and/or mixtures of solvents may be identified by routine experimentation. A preferred organic solvent is n-butanol.
Calculating the volume of organic solvent needed to extract the acidic lipopeptide antibiotic chelate from aqueous solution is well within the routine capabilities of skilled artisans. Typically, the volume of the organic solvent ranges about ⅓to about 3 times the volume of the aqueous solution. Preferably, the volume of organic solvent used to extract the acidic lipopeptide antibiotic chelate from aqueous solution is about equal to the volume of the aqueous solution.
The aqueous solution may be contacted with as many portions of organic solvent necessary to extract substantially all of the acidic lipopeptide antibiotic chelate into organic solvent. The number of portions of organic solvent necessary to completely extract the acidic lipopeptide antibiotic chelate from aqueous solution (generally, at least two portions of the organic solvent will be used to extract the lipopeptide antibiotic) may be readily determined by one of skill in the art.
Generally, contacting an aqueous solution containing an acidic lipopeptide antibiotic chelate with organic solvent in a separatory funnel is sufficient to extract the acidic lipopeptide antibiotic chelate into organic solvent. However, in some situations, the organic solvent and aqueous solution may be contacted by other methods well known to the skilled artisan such, as by magnetic stirring, mechanical stirring, sonication, etc. Further, in some situations (i.e., in scale-up procedures) continuous liquidxe2x80x94liquid extraction may be used to extract the acidic lipopeptide antibiotic chelate into organic solvent.
The acidic lipopeptide antibiotic chelate may be disrupted by contacting organic solvent containing the chelate with acid. Preferably, the organic solvent containing the acidic lipopeptide antibiotic metal chelate is contacted with an aqueous acid solution, most preferably, with aqueous mineral acid. Ideally, the pH of the aqueous acid solution is sufficiently acidic to completely protonate the carboxylate groups of the acidic lipopeptide antibiotic chelate. Alternative methods of protonating carboxylate groups in organic solvent are known to the skilled artisan (e.g., saturating organic solvents with gaseous acids such as gaseous HCl or gaseous HBr, use of strong organic acids, etc.). Preferably, the pH of the aqueous acid solution is between about 3.0 and about 1.0, more preferably, between about 2.5 and about 1.5 and most preferably, is about 2.0. While not wishing to be bound by any particular theory of operation, it is believed that acidifying the organic phase protonates the carboxylate groups of the lipopeptide antibiotic chelate, thereby disrupting metal chelation by the carboxylic acid.
Once the organic phase containing the acidic lipopeptide antibiotic chelate has been acidified, the acidic lipopeptide antibiotic, which now behaves as a conventional carboxylic acid, may be partitioned between organic solvent and aqueous acid and base solutions by methods known to those of skill in the art. Thus, for example, the organic solvent containing the free acid of the lipopeptide antibiotic may be contacted with aqueous base solution to provide an aqueous extract of a salt of the lipopeptide antibiotic. Then, treatment of the aqueous solution of the salt of the lipopeptide antibiotic with aqueous acid solution allows extraction of the free acid of the lipopeptide antibiotic into organic solvent. The free acid of the lipopeptide antibiotic after treatment with neutral or basic aqueous solution, may be again extracted into aqueous solution by conversion to the salt of the lipopeptide antibiotic. The acidic lipopeptide antibiotic may be isolated as either the salt or the free acid using methods known to the skilled artisan. The isolated acidic lipopeptide antibiotic may, if desired, be further purified by conventional methods well known to the skilled artisan such as silica gel chromatography, ion exchange chromatography, reverse phase chromatography, etc.